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Improving biosecurity of bovine in vitro embryo production and cryopreservationMoreira, Vanessa. January 2009 (has links)
Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on June 17, 2009). Includes bibliographical references.
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Improving biosecurity of bovine in vitro embryo production and cryopreservationMoreira, Vanessa January 2009 (has links)
The experiments reported in this thesis were conducted to overcome limitations in conventional processes of <i>in vitro-</i>production of embryos and to develop novel procedures for cryopreservation of bovine oocytes and embryos. The experiments, concentrating initially on unfertilized oocytes and subsequently on embryos, used methods relevant to livestock reproductive biotechnology. Key aims were to overcome reliance on media using animal-derived constituents such as serum and albumin. Bovine oocytes matured <i>in vitro</i> in a novel biosafe formulation (MM1mat) excluding serum and albumin reached blastocyst stages (after <i>in vitro</i> fertilization) at rates equivalent to those achieved with conventional protocols (P>0.05). Experiments that investigated development of bovine zygotes in an original series of protein-free culture media (V1 for embryos to Day 4 post-fertilization; V2a and similar formulations for Day 4 onwards) showed that sequential culture in V1/V2a did not compromise development to the blastocyst stage when compared to ‘monoculture’ in an albumin-supplemented medium (P>0.05). Blastocysts produced in V1/V2a survived better after vitrification than counterparts produced in presence of albumin. Vitrification solutions (also often reliant on harmful serum-supplemented media) could be superseded by novel alternatives using V2a as a biohazard-free base medium. It was concluded that bovine blastocysts can be produced and cryopreserved safely in the novel media. A further important aspect of the investigations was that they developed a new biosecure ‘closed system’ for cryopreservation of oocytes and embryos. That system employed the CryoTip –which could be sealed before contact with liquid nitrogen – and the subsequent metabolic and biochemical studies on V1/V2a-produced blastocysts, either fresh or vitrified, indicated pyruvate metabolism, amino acids depletion and peroxide status parameters were equivalent to controls.
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Adição de proteína plasmática a associada à prenhez (PAPP-A) durante a maturação in vitro aumenta o IGF-1 biodisponível e modula o perfil transcricional de complexos cumulus-oócitos e embriões bovinos / Addition of pregnancy-associated plasma protein-A (PAPP-A) during in vitro maturation increases bioavailable IGF-1 and modulates transcriptional profile of bovine cumulus-oocyte complexes and blastocystsGiroto, Alan Brunholi 25 May 2018 (has links)
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Previous issue date: 2018-05-25 / In this study we aimed to evaluate how PAPP-A addition during in vitro maturation affected the IGF-1 quantification, transcript abundance related to cumulus oocyte complexes (COCs) and blastocysts quality, embryonic yield, as well as post-warming survival. We matured COCs through a 24 h treatment of TCM199 serum-free medium, either with PAPP-A supplementation (100 ng/mL; PAPP-A group) or without (control). Maturation medium was collected for IGF-1 quantification, and matured COCs were used for in vitro fertilization and culturing. The PAPP-A group exhibited 1.27 times higher IGF-1 concentrations than control. A comparison of in vitro embryo production across the groups found no difference in cleavage rate, embryonic yield, and survival, 3 and 24 h post-cryopreservation. In PAPP-A oocytes, only TXNRD1 was up-regulated. However, in PAPP-A cumulus cells, VNN1 and HDAC2 were up-regulated, while AGPAT1, AGPAT9, FASN, CASP3, EGFR, HAS2, IMPDH1, and MTIF3 were down-regulated. Finally, in PAPP-A blastocysts, CPT2, CASP9, DNMT3A, TFAM, and KRT8 were up-regulated, while ATF4, CASP3, and IFITM3 were down-regulated. We concluded that PAPP-A addition increased IGF-1 but did not influence embryonic yield and survival. Nevertheless, elevated IGF-1 could improve embryo competence through modulating expression of genes involved with lipid metabolism, oocyte competence and apoptosis in COCs and blastocysts. / A protéina sérica associada à prenhez (PAPP-A) é capaz de modular a biodisponibilidade do IGF-1 (fator de crescimento semelhante à insulina tipo 1) pela quebra das ligação com as proteínas de ligação do IGF (IGFBPs). O objetivo foi avaliar como a adição da PAPP-A durante a maturação in vitro (MIV) afetou a biodisponibilidade de IGF-1, a abundância de transcritos nos oócitos e células do cumulus e blastocistos, a produção embrionária e a sobrevivência pós aquecimento. Foi realizada a MIV de 24 h em complexos cumulus oócitos (CCOs – 20/grupo) oriundos de abatedouro, em meio TCM199 livre de soro, com adição de PAPP-A (100 ng/mL; grupo PAPP-A) ou sem (controle). O IGF-1 foi quantificado no meio de maturação e os CCOs maturados foram utilizados na fertilização e cultivo in vitro. Oócitos e células do cúmulus foram separados; e blastocistos foram congelados para a realização da expressão gênica. Taxa de clivagem e produção de blastocistos foram calculados como porcentagem e transformados para arco seno. Dados de expressão gênica foram normalizados com a média do grupo de controle. Os resultados foram analisados por test t, porém a criopreservação embrionária foi testada por Qui-quadrado (JMP software, SAS). Diferenças foram consideradas significativas quando p ≤ 0,05. O grupo PAPP-A apresentou 27% mais concentração de IGF-1 biodisponível. Na produção in vitro de embriões, não encontrou-se diferença na taxa de clivagem, produção e sobrevivência embrionária. Apenas o gene TXNRD1 mostrou maior expressão nos oócitos do grupo PAPP-A. No entanto, nas células do cumulus PAPP-A, o VNN1 e HDAC2 apresentaram maior expressão, enquanto os genes AGPAT1, AGPAT9, FASN, CASP3, EGFR, HAS2, IMPDH1 e MTIF3 apresentaram menor expressão. Nos blastocistos PAPP-A, os genes CPT2, CASP9, DNMT3A, TFAM e KRT8 apresentaram maior expressão, enquanto os genes ATF4, CASP3 e IFITM3 apresentaram menor expressão. Em conclusão, a adição de PAPP-A durante a MIV aumentou o IGF-1 biodisponível, mas não influenciou a produção e a sobrevivência embrionária após desvitrificação. No entanto, o aumento do IGF-1 biodisponível pode melhorar a competência embrionária através da modulação da expressão gênica em oócitos, células do cúmulus e blastocistos.
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